Proteins are labeled with radioactive halogen isotopes for both in vitro and in vivo study and clinical purposes. Radiohalogenated proteins are used, for example, for in vivo investigation of protein metabolism and pharmacokinetics as well as a tool for radioimmunoassays. Antibodies are labeled with halogen radioisotopes for diagnostic imaging and radiotherapy. The following table shows the applications of various halogen isotopes:
______________________________________ Half-life Type of Energy Isotope (hours) Application emission (keV) ______________________________________ .sup.18 F 1.83 diagnostic, PET positron 511 .sup.75 Br 1.63 diagnostic, PET positron 511 .sup.76 Br 16.1 diagnostic, PET positron 511 .sup.77 Br 57.0 diagnostic gamma 239 therapeutic Auger &lt;5 .sup.123 I 13.0 diagnostic gamma 159 .sup.131 I 193 diagnostic gamma 364 therapeutic beta 607 .sup.211 At 7.2 therapeutic alpha 6786 ______________________________________
The development of highly specific monoclonal antibodies which can localize in cancerous tissue has increased the utility of radiohalogenated antibodies for diagnosis and therapy.
Therefore, methods have been sought to attach radiohalogens to protein molecules so that the labeled molecule remains stable to decomposition in vivo without disrupting the structure or biological function of the protein.
The strength of the aromatic carbon-halogen bond relative to that of the aliphatic carbon-halogen bond has led to the development of methods for attaching halogens to proteins through aryl-halogen bonds. Proteins may be halogenated directly or the protein may be conjugated to a separately halogenated compound.
Direct halogenation involves the substitution of halogen for a hydrogen atom on the amino acid residue of the protein. For example, a tyrosine residue may be radioiodinated ortho to the hydroxy group by the reaction of sodium iodide in the presence of an oxidizing agent at neutral pH. Other amino acid residues such as histidines, tryptophans and methionines may also be iodinated depending on reaction conditions. Common oxidizing agents for iodination of proteins are Iodogen (1,3,4,6-tetrachloro-3.alpha.,6.alpha.-diphenylglycoluril), N-bromosuccinimide and Chloromine-T (sodium N-chloro-p-toluenesulfonamide). The use of oxidizing agents for direct radioiodination of proteins, however, causes denaturation and/or chemical alteration of the binding site, reducing the biological activity of the protein. Therefore, new conjugation methods for radiohalogenation have been sought.
Conjugation involves the radiohalogenation of a small molecule which is then coupled to the protein by an acylation reaction. Bolton and Hunter first reported the now most commonly used conjugation method in their article, "The Labelling of Proteins to High Specific Radioactivities by Conjugation to a .sup.125 I-Containing Acylating Agent" 133 Biochem. J. 529-539 (1973). According to the Bolton-Hunter procedure, 3-(4-hydroxypheny 1) propionic acid N-hydroxysuccinimide ester is radioiodated ortho to the hydroxy group with Chloromine-T to obtain a compound of the formula, ##STR3## which reacts with the free amino groups of proteins to form an amide bond. The principal limitation of the Bolton-Hunter method is that only 30-40% of the radioactive iodine is coupled to the protein due to rapid hydrolysis of the active ester in aqueous solution. Use of the Bolton-Hunter reagent does have the advantage that more of the biological function of the protein is retained than when proteins are directly radioiodinated.
Wood et al. reported another radioiodination method in their article, "The Radioactive Labeling of Proteins with an Iodinated Amidination Reaction", 69 Analytical Biochemistry 339-349 (1975). The Wood method involves the Chloromine-T radioiodination of methyl-p-hydroxybenzimidate hydrochloride ortho to the hydroxy group to produce a compound of the formula, ##STR4## which reacts with protein amino groups to form an amidine linkage. Use of the Wood reagent also avoids the direct contact of deleterious oxidizing agents with the protein; however, yields of radioiodinated compound are less than 20%, even after a 24 hour reaction period.
Another major problem with the Bolton-Hunter and Woods reagents is in vivo loss of radioiodine from the protein. Unassociated radioiodine accumulates in the thyroid and stomach and appears in the urine after administration of radioiodinated antibodies. Dehalogenation is believed to occur in vivo by hydrolytic and enzymatic processes as well as those mediated by binding of the protein to a cell. The Bolton-Hunter and Woods methods involve radiohalogenation of a phenyl ring which is activated by a hydroxy group, causing substitution at the ortho position. The presence of the hydroxy group ortho to the radiohalogen, however, renders the conjugate susceptible to both hydrolytic and enzymatic in vivo dehalogenation. Therefore, radiohalogenated conjugates which do not have a hydroxy group ortho to the halogen label on the phenyl ring have been sought.
Wilbur et al. European Patent 0 203 764 discloses substitution of radiohalogen on a non-activated aromatic ring for conjugation to proteins. According to Wilbur et al., a haloaryl compound is initially converted into an aryllithium compound, which, upon transmetalation with R.sub.3 SnCl (R=n--C.sub.4 H.sub.9 or CH.sub.3), HgX.sub.2, BX.sub.3 (X=halogen), or BZ.sub.3 (Z=alkyl or alkoxy), gives an appropriate organometallic derivative of Sn, Hg or B. Such organometallic derivatives are then utilized for site-specific radiohalogenation. The Wilbur et al. aryl compounds also have a non-activating short-chain substituent bearing a functional group suitable for conjugation to protein under conditions that preserve the biological activity of the protein.
Applicants have reported the radioiodination of proteins using the iodinated derivative, N-succinimidyl 3-(tri-n-butylstannyl) benzoate (Activated Tin-containing Ester, hereinafter "ATE") having the formula, ##STR5## Zalutsky et al., "A Method for the Radiohalogenation of Proteins Resulting in Decreased Thyroid Uptake of Radioiodine", 38 Appl. Radiat. Isot. 1051-1055 (1987). Radioiodination of ATE achieves 80% yield and proteins are coupled with the resulting labeled compound with greater than 60% efficiency. In vivo dehalogenation of [.sup.125 I]ATE-labeled goat IgG is reduced compared with dehalogenation goat IgG labeled using the Iodogen method.